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Structure, folding and interactions of membrane-associated biomolecules studied by NMR


Mareš, J. Structure, folding and interactions of membrane-associated biomolecules studied by NMR. 2009, University of Zurich, Faculty of Science.

Abstract

In the course of my doctoral studies I focused on the characterization of the
structure and folding of membrane-interacting molecules such as the neuropeptide
peptide YY (PYY), glycolipids derived from gp120, or polymyxins and their
interactions with the membrane-surface components such as lipopolysaccharides
(LPS). Studies of structure and folding were caried out on PYY, a small peptide for
which membrane association was postulated by us previously, whereas the study on
the glycolipid-cyanovirin and polymyxin-lipopolysaccharid systems focused on the
interaction between peptides/proteins and carbohydrates.
Some of the peptides from the neuropeptide Y family are know to adopt a
relatively stable and well-defined helical hairpin structure in water. The characteristic
hairpin formed by a polyproline helix associated with an α-helix results in a particular
type of teriary structure known also as the PP-fold. The latter was first determined by
X-ray crystallographic analysis of the avian pancreatic peptide (aPP). In this work we
have investigated the significance of contacts between resuides from the α-helix and
the polyproline helix for forming tertiary structure. In order to do so we redetermined
the structure of PYY in water using an extended set of NOEs complemented by
RDCs. These data helped to significantly increase the resolution of the structure, and
to propose details important for the particular nature of tertiary interactions. The most
remarkable improvement was obtained for the turn region linking these two helices.
The tertiary interactions are formed by specific Tyr-Pro contacts, and similar contacts
are observed in other peptides of the family, that form the characteristic PP-fold.
Furthermore, the importance of these interactions was analysed by substituting them
via site-directed mutagenesis. Subsequent characterization of their dynamics was
carried out by measuring heteronuclear 15N{1H}-NOE data, as well as chemical shift
changes during thermal and solvent induced (un)folding of wild type PYY. The
unfolding was followed by tracking the changes of the C-α chemical shifts. The
thermal denaturation revealed concerted changes occurring at all atoms observed, and
according to the cooperative nature of the changes, classified PYY as a two-state
folder. The solvent denaturation was performed by stepwise changing the solvent
system from pure water to pure methanol in 10% intervals, while the other conditions
were kept constant. In previous work of our group, methanol was shown to mimic
well the environment at the water-membrane interface. As an example, it was shown,
that the structure of PYY in DPC micelles lacked tertiary contacts and is very similar
to the one present in methanol. Therefore, the methanol denaturation resembles
structural changes accompanying increasing participation in membrane-surface
contacts. This study determined the concentration at which the tertiary structure
denatures, but it also revealed rigidification of secondary structures at high methanol
concentrations. The complex methanol unfolding curves also revealed a dramatic
alteration of proline isomerization dynamics.
The second part of my doctoral work focused on studying a glycolipid, derived
from the carbohydrate portion of the HIV glycoprotein gp120, incorporated into DPC
micelles as a membrane model. In order to validate recognition of this memraneincorporated
glycolipid model, its interaction with a carbohydrate-recognizing protein
was studied by high-resolution NMR spectroscopy. In cooperation with Prof. Schmidt
from the University of Konstanz a terminal arm of the high-mannose structure of the
HIV surface-envelope protein gp120 was synthesized as the mono- di- and trimannose
moiety linked to an aliphatic membrane anchor. I have then characterized
I I
the insertion of this glycolipid into DPC micelles using spin-labels and by measuring
the changes in translation diffusion rates by NMR techniques.
Cyanovirin (CV-N) is cyanobacterial lectin, which binds the above-mentioned
high-mannose arm of gp120, thereby preventing HIV virus cell entry. A 15N
isotopically labeled CV-N was used in chemical shift mapping studies to monitor its
interaction with the micelle-anchored glycolipid. The results proved that CV-N
recognizes properly the micelle-anchored glycolipid. Using DPC-incorporated spin
labels, it was further confirmed that upon CV-N binding the glycolipid remains
anchored into the DPC micelles. It could be shown, that binding occurs at the same
sites as for the free dimannoside. In contrast to hydrophobic tertiary interactions of
PYY, these interactions include polar groups, which were not disrupted by the
membrane environment. Additionally, upon competition with the better ligand
trimannoside, we detected detachment of the DPC-micelle-bound CV-N. We envisage
that this system may become useful for the reversible attachment of biomolecules to
membrane surfaces.
Lipopolysaccharide (LPS) forms the main constituent of the outer membrane of
Gram-negative bacteria. LPS is also known for its toxic effects. Septic shock, with a
mortality rate of about 50%, is a result of hyperactivation of the immune system and
is caused in 70% of cases by LPS. This negatively charged molecule inevitably
interacts with cationic antimicrobial peptides, such as polymyxins (PMXs). The
strong interaction leads to disruption of the integrity of the outer membrane and may
suppress the septic shock initiated by LPS molecules released from disrupted bacterial
membranes. In the last part of this thesis work we investigated the LPS-polymyxin
interaction. We have chosen a bacterial strain producing a simple form of LPS. A
purification method was developed that consisted of an extraction protocol, used
before, and a new HPLC chromatography step in combination with an optimized
ternary solvent mixture. This procedure facilitated production of the isotopically
labeled and chemically defined compound in high purity. Various techniques of
heteronuclear NMR spectroscopy allowed observing its insertion in DPC micelles.
Furthermore, characterization of the LPS-polymyxin interaction was performed using
commercially available polymyxin-B and -E, as well as polymyxin-M (PMX-M),
which was expressed, isolated and purified in house. Chemical shift mapping was
performed by measuring carbon-proton HSQC experiments of isotopically labeled
LPS and unlabeled peptides. In addition, in a complementary experiment isotopically
labeled polymyxin-M and unlabeled LPS were used. The data enabled us to localize
the interaction sites between LPS and polymyxins. Additional information was
derived from isotope-filtered NOESY experiments using 13C-labeled LPS and
unlabeled polymyxins. Since the signals of the majority of atoms involved in the
intermolecular interaction cannot be observed due to linebroading caused by
exchange effects, NOESY experiments did not provide sufficient information for the
determination of the LPS-PMX complex at reasonable detail. We have employed a
combination of simulated annealing and molecular dynamics calculation to determine
the possible structure of the LPS-PMX complex in presence of micelles. The
simulated annealing utilized sparse experimental restrains derived from the isotopefiltered
NOESY and generated a large set of conformers. Analysis of the obtained set
allowed selection of those structures, where the intermolecular contacts were in
agreement with the chemical shift mapping patterns. Further refinement and analysis
was performed by molecular dynamics calculation. Both solvent (water) and
cosolvent (DPC) molecules were explicitly included in the calculation. In this work,
II I
we have prepared and characterized all the constituents of the complex biological
interaction, proposed the structure of the complex and characterized the nature of the
interacting moieties.

Abstract

In the course of my doctoral studies I focused on the characterization of the
structure and folding of membrane-interacting molecules such as the neuropeptide
peptide YY (PYY), glycolipids derived from gp120, or polymyxins and their
interactions with the membrane-surface components such as lipopolysaccharides
(LPS). Studies of structure and folding were caried out on PYY, a small peptide for
which membrane association was postulated by us previously, whereas the study on
the glycolipid-cyanovirin and polymyxin-lipopolysaccharid systems focused on the
interaction between peptides/proteins and carbohydrates.
Some of the peptides from the neuropeptide Y family are know to adopt a
relatively stable and well-defined helical hairpin structure in water. The characteristic
hairpin formed by a polyproline helix associated with an α-helix results in a particular
type of teriary structure known also as the PP-fold. The latter was first determined by
X-ray crystallographic analysis of the avian pancreatic peptide (aPP). In this work we
have investigated the significance of contacts between resuides from the α-helix and
the polyproline helix for forming tertiary structure. In order to do so we redetermined
the structure of PYY in water using an extended set of NOEs complemented by
RDCs. These data helped to significantly increase the resolution of the structure, and
to propose details important for the particular nature of tertiary interactions. The most
remarkable improvement was obtained for the turn region linking these two helices.
The tertiary interactions are formed by specific Tyr-Pro contacts, and similar contacts
are observed in other peptides of the family, that form the characteristic PP-fold.
Furthermore, the importance of these interactions was analysed by substituting them
via site-directed mutagenesis. Subsequent characterization of their dynamics was
carried out by measuring heteronuclear 15N{1H}-NOE data, as well as chemical shift
changes during thermal and solvent induced (un)folding of wild type PYY. The
unfolding was followed by tracking the changes of the C-α chemical shifts. The
thermal denaturation revealed concerted changes occurring at all atoms observed, and
according to the cooperative nature of the changes, classified PYY as a two-state
folder. The solvent denaturation was performed by stepwise changing the solvent
system from pure water to pure methanol in 10% intervals, while the other conditions
were kept constant. In previous work of our group, methanol was shown to mimic
well the environment at the water-membrane interface. As an example, it was shown,
that the structure of PYY in DPC micelles lacked tertiary contacts and is very similar
to the one present in methanol. Therefore, the methanol denaturation resembles
structural changes accompanying increasing participation in membrane-surface
contacts. This study determined the concentration at which the tertiary structure
denatures, but it also revealed rigidification of secondary structures at high methanol
concentrations. The complex methanol unfolding curves also revealed a dramatic
alteration of proline isomerization dynamics.
The second part of my doctoral work focused on studying a glycolipid, derived
from the carbohydrate portion of the HIV glycoprotein gp120, incorporated into DPC
micelles as a membrane model. In order to validate recognition of this memraneincorporated
glycolipid model, its interaction with a carbohydrate-recognizing protein
was studied by high-resolution NMR spectroscopy. In cooperation with Prof. Schmidt
from the University of Konstanz a terminal arm of the high-mannose structure of the
HIV surface-envelope protein gp120 was synthesized as the mono- di- and trimannose
moiety linked to an aliphatic membrane anchor. I have then characterized
I I
the insertion of this glycolipid into DPC micelles using spin-labels and by measuring
the changes in translation diffusion rates by NMR techniques.
Cyanovirin (CV-N) is cyanobacterial lectin, which binds the above-mentioned
high-mannose arm of gp120, thereby preventing HIV virus cell entry. A 15N
isotopically labeled CV-N was used in chemical shift mapping studies to monitor its
interaction with the micelle-anchored glycolipid. The results proved that CV-N
recognizes properly the micelle-anchored glycolipid. Using DPC-incorporated spin
labels, it was further confirmed that upon CV-N binding the glycolipid remains
anchored into the DPC micelles. It could be shown, that binding occurs at the same
sites as for the free dimannoside. In contrast to hydrophobic tertiary interactions of
PYY, these interactions include polar groups, which were not disrupted by the
membrane environment. Additionally, upon competition with the better ligand
trimannoside, we detected detachment of the DPC-micelle-bound CV-N. We envisage
that this system may become useful for the reversible attachment of biomolecules to
membrane surfaces.
Lipopolysaccharide (LPS) forms the main constituent of the outer membrane of
Gram-negative bacteria. LPS is also known for its toxic effects. Septic shock, with a
mortality rate of about 50%, is a result of hyperactivation of the immune system and
is caused in 70% of cases by LPS. This negatively charged molecule inevitably
interacts with cationic antimicrobial peptides, such as polymyxins (PMXs). The
strong interaction leads to disruption of the integrity of the outer membrane and may
suppress the septic shock initiated by LPS molecules released from disrupted bacterial
membranes. In the last part of this thesis work we investigated the LPS-polymyxin
interaction. We have chosen a bacterial strain producing a simple form of LPS. A
purification method was developed that consisted of an extraction protocol, used
before, and a new HPLC chromatography step in combination with an optimized
ternary solvent mixture. This procedure facilitated production of the isotopically
labeled and chemically defined compound in high purity. Various techniques of
heteronuclear NMR spectroscopy allowed observing its insertion in DPC micelles.
Furthermore, characterization of the LPS-polymyxin interaction was performed using
commercially available polymyxin-B and -E, as well as polymyxin-M (PMX-M),
which was expressed, isolated and purified in house. Chemical shift mapping was
performed by measuring carbon-proton HSQC experiments of isotopically labeled
LPS and unlabeled peptides. In addition, in a complementary experiment isotopically
labeled polymyxin-M and unlabeled LPS were used. The data enabled us to localize
the interaction sites between LPS and polymyxins. Additional information was
derived from isotope-filtered NOESY experiments using 13C-labeled LPS and
unlabeled polymyxins. Since the signals of the majority of atoms involved in the
intermolecular interaction cannot be observed due to linebroading caused by
exchange effects, NOESY experiments did not provide sufficient information for the
determination of the LPS-PMX complex at reasonable detail. We have employed a
combination of simulated annealing and molecular dynamics calculation to determine
the possible structure of the LPS-PMX complex in presence of micelles. The
simulated annealing utilized sparse experimental restrains derived from the isotopefiltered
NOESY and generated a large set of conformers. Analysis of the obtained set
allowed selection of those structures, where the intermolecular contacts were in
agreement with the chemical shift mapping patterns. Further refinement and analysis
was performed by molecular dynamics calculation. Both solvent (water) and
cosolvent (DPC) molecules were explicitly included in the calculation. In this work,
II I
we have prepared and characterized all the constituents of the complex biological
interaction, proposed the structure of the complex and characterized the nature of the
interacting moieties.

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Additional indexing

Item Type:Dissertation
Referees:Zerbe O, Robinson J
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2009
Deposited On:08 Jan 2010 13:35
Last Modified:06 Dec 2017 22:42
Number of Pages:182
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&con_lng=GER&func=find-b&find_code=SYS&request=005872128

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